Escherichia coli has been generally used as a host in genetic engineering. However, the use of E. coli has many disadvantages such as that peptides or proteins produced by recombined genes would not be secreted into media but remain within the cells, so that their production will be naturally limited, and therefore that the extraction and purification of the peptides or proteins involve complicated procedures such as cell grinding and affinity chromatography.
Udaka et al. studied to find microorganisms capable of secreting proteins out of the cells as hosts for genetic engineering, and consequently isolated and identified five strains including four belonging to B. brevis and one belonging to Bacillus proteiformans each secreting a large amount of proteins, among approximately 1,200 strains (cf. Agric. Biol. Chem., 40 (3), 523-528 (1976)).
Also B. subtilis is used as a secreting host and used to obtain accumulation of various heterologous proteins including .alpha.-amylase and interferon. However these attempts do not necessarily give satisfactory results, since proteases present inside or outside the cells would restrict the amount of the products or even decompose the same.
Previously Udaka et al. found that B. brevis 47, which harbored pBAM101 obtained by introducing a thermostable .alpha.-amylase gene of Bacillus stearothermophilus DY-5 into plasmid pUB110, and B. subtilis harboring pBAM101 produced approximately 15,000 U/ml and 3,000 U/ml of .alpha.-amylase in media respectively when cultured at 37.degree. C. for 48 hours (cf. J. Bacteriol., 164 (3), 1182-1187 (1985)).
Thus it has been proved that a heterologous gene product can be efficiently produced by utilizing the protein-secreting ability of a protein-producing bacterium, B. brevis 47, since B. brevis 47, which will be described in detail hereinbelow, can produce a thermostable .alpha.-amylase approximately five times as much as B. subtilis, having the same plasmid as that in the former, does.
However, all of the above-mentioned five strains, each isolated as a bacterium capable of secreting a large amount of protein out of the cells, i.e., B. brevis 47, 144, 481 and 899 and B. proteiformans 444, would decompose bovine serum albumin, which will be abbreviated as BSA hereinafter, when grown in a medium containing BSA. In addition, it has been proved that B. brevis 144, 481 and 899 and B. proteiformans 444 are further capable of decomposing casein. These facts suggest that peptides and proteins efficiently produced by these bacteria, each employed as a host capable of secreting a large amount of protein out of the cells in the production of a heterologous gene product with the use of a recombined gene, may be decomposed with protease.